Biochemical studies make extensive use of static chromatographies such as high resolution two dimensional paper and thin layer chromatography and electrophoresis. The information available from such techniques is limited by the relatively poor performance of the detection systems traditionally used. This proposal supports the development of a mass spectrometric system which can analyze separated biomolecules in situ, with a particular focus on the analysis of peptide mixtures separated by high performance two dimensional electrophoresis. With information available from the mass spectrum, the identity and homogeneity of the separated components can be established without loss of the integrity of the chromatogram. In favorable instances, the compounds can be recovered after analysis for further characterization or processing. In this new instrument, intact chromatograms are placed under vacuum, and components encapsulated in the matrix are brought into focus by movement of the chromatogram in the xy development plane. A secondary ion mass spectrometer with a highly focused primary ion beam sputters material directly from the chromatogram into a quadrupole mass analyzer. A spatial resolution of 10 microns in both x and y can increase the effective resolution of poorly separated compounds by processing of the mass spectral data. A feedback system automatically maximizes the efficiency of data collection by increasing spatial resolution as compounds are brought into the instrument focus. Initial applications will be to the analysis of carnitine-based samples separated by thin layer chromatography, to sickle cell hemoglobin peptides separated by electrophoresis, and to a new experiment, selected sequence monitoring, which pinpoints peptides containing a specific sequence of amino acids from within a mixture separated by two dimensional chromatography. These initial experiments document the performance of the new system in problems which have significant biochemical interest. By the end of the proposed period, an automated analysis system will have been developed which can be used for general analysis of complex mixtures separated by two dimensional electrophoresis.